We addressed the question of whether glial cells in intact white matter tracts express neurotransmitter receptors and we used Ca++ signalling as a probe to detect the receptor activation. Corpus callosum slices from postnatal mice were bulk‐loaded with the Ca++‐ sensitive fluorescent dye fluo‐3, and confocal microscopy was used to measure Ca++ transients in response to neuroligands. Glial cell bodies were intensely dye‐loaded and could be discriminated from the diffuse fluorescence of axons. Subpopulations of glial cells from slices obtained at postnatal days 3 to 7 responded with Ca++ signals to ATP, glutamate, histamine, GABA, norepinephrine, serotonin, angiotensin II, bradykinin, and substance P. These subpopulations showed a distinct overlap; cells which were responsive to substance P always showed Ca++ signalling in response to histamine, ATP, GABA, and high K+ (membrane depolarization). GABA‐responsive cells almost always showed a [Ca++], increase after membrane depolarization. In brain slices from postnatal day 11 to 18 animals, the Ca++ responses were evident for glutamate, ATP, and norepinephrine, while GABA, substance P, serotonin, histamine, or angiotensin II rarely elicited a response. This study demonstrates that white matter glial cells in slices exhibit a large repertoire of neurotransmitter responses linked to Ca++ signalling and that these receptor systems are differentially distributed on sub‐populations of glial cells.